J. Mol. Biol. (1990) 213, 79-108

R N A Chain Elongation by Escherichia coli R N A Polymerase Factors Affecting the Stability of Elongating Ternary Complexes Karen M. Arndt and Michael J. Chamberlin

Department of Biochemistry University of California Berkeley, CA 94720, U.S.A. (Received 13 January 1989; accepted 22 November 1989) We have devised a method to follow the stability of individual ternary transcription complexes containing Escherichia coli RNA polymerase halted at many different sites along a DNA template during the transcription process. Studies of complexes formed with phage T7 DNA templates reveal at least three general classes of ternary complexes that differ dramatically in their properties. Complexes of one sort (normal complexes) are highly stable to dissociation and denaturation under a variety of solution conditions. They remain intact and active for up to 24 hours even in salt concentrations up to 1 M-K +. This suggests that they are stabilized to a significant extent by non-ionic interactions between RNA polymerase and the nucleic acids. We consider these to be the normal complexes formed during RNA chain elongation. Complexes of a second sort (release complexes) dissociate rapidly, releasing free RNA transcripts and active RNA polymerase. The rate of dissociation is substantially enhanced by elevated concentrations of K +, hence the interaction between RNA potymerase and nucleic acids in these complexes is stabilized predominantly by ionic interactions. However, release complexes are stabilized by millimolar concentrations of Mg 2+, which has been implicated in stabilization of the binding of RNA to free RNA polymerase. These complexes are formed at DNA sequences that we refer to as release sites. Analysis of DNA sequences at release sites reveals that all share a common feature, the potential to form an RNA hairpin in the region just upstream from the actual 3' end of the released RNA. Experiments incorporating IMP in the transcript and blocking potential hairpin formation with DNA oligomers support a direct role for an RNA hairpin in triggering the release reaction. Changes in the 3'-proximal DNA sequences generally have little effect on the presence or rate of the release reaction, although there are significant exceptions. The results suggest that the presence of certain RNA hairpins in the region six to ten nucleotides upstream from the transcript growing point can trigger a substantial structural transition in the ternary transcription complex, forming a "release mode" complex from which transcript dissociation is facilitated. This release, mode complex may be a central intermediate in RNA chain termination. A final class of complexes (dead-end complexes) appear to be elongating complexes that have entered a state or conformation that is stable, but is blocked in resuming the normal elongation reaction. Such complexes bear active RNA polymerase, and can be restarted after limited pyrophosphorolysis. The structural elements that determine the formation of dead-end complexes are not yet known.

1. Introduction

controlled by interaction of the enzyme in such a ternary transcription complex with sequences in the DNA and RNA, and with regulatory factors that control pausing, termination and antitermination. In both prokaryotes and eukaryotes there is ample evidence that transcription is regulated through termination, pausing and antitermination processes that often couple the synthesis of a transcript to its continued utilization (Platt, 1986; Friedman et al.,

Initiation of transcription leads to formation of stable ternary complexes that contain template DNA, RNA polymerase and nascent transcript. This process leads to a commitment of the RNA polymerase to a particular DNA template molecule that is relieved only when transcript and polymerase are released in the termination process. Passage of the RNA polymerase along the DNA is 0022-2836/90/090079-30 $03.00/0

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K. M. A r n d t and M. J. Chamberlin

1987; Yanofsky, 1988). These regulatory mechanisms depend on the unique features of particular ternary transcription complexes located at different points along the transcription unit. This fact is most evident for the complexes formed at termination sites, since they dissociate to release enzyme and transcript. Similarly, there are several known examples of sites that lead to transcriptional pausing that are believed to play a role in cellular regulation (Winkler & Yanofsky, 1981; Turnbough et al., 1983; Navre & Schachman, 1983). Some antitermination processes must also depend on special properties of ternary complexes: loading of the lambda phage N protein is dependent on specific nut sites in the transcription unit that are encountered during elongation, and on successful interaction of the host nus proteins with the transcribing RNA polymerase (Friedman et al., 1987; Barik et al., 1987). In one case, interaction of a protein factor with RNA polymerase at a nut site may convert this site to a terminator (Robert et al., 1987). I n vitro studies using purified bacterial RNA polymerases reinforce this general picture. Different DNA sequences can lead to transcriptional pausing and termination (Yager & yon Hippel, 1987). These events can be suppressed or enhanced, in a sequence-dependent manner, by protein factors or small molecules that interact directly with the RNA polymerase protein. From these considerations it seems evident that the structure and properties of ternary complexes must be quite distinct at different points along a transcription unit. Studies of mixed populations of ternary complexes have shown that ternary complexes are generally stable at elevated salt concentrations (Richardson, 1966; Fuchs et al., 1967; Fukuda & Ishihama, 1971; Beabealashviily & Savotchkina, 1973). Similar studies have measured the average size of the DNA fragments protected from DNase I digestion of ternary complexes (Beabealashvilly et al., 1972; Rohrer & Zillig, 1977) and the average unwinding angle per RNA polymerase (Gamper & Hearst, 1982; Amouyal & Buc, 1987). Unfortunately, many reviewers have assumed that normally elongating ternary complexes all have structures resembling these average values, a conclusion that can be questioned in light of our earlier discussion. There have been few studies of ternary complexes positioned at specific sites (Carpousis & Gralla, 1985; Straney & Crothers, 1987; Levin et al., 1987; Landick & Yanofsky, 1987), and these studies have not allowed comparative evaluation of the effect of different sequences on the structure and properties of such complexes. The importance of understanding the effect of different DNA sequences on the properties of ternary transcription complexes, and on their interactions with regulatory factors, has led us to search for general methods to prepare and study such complexes (Levin et al., 1987). We describe here studies employing an approach that allows charac-

terization of the properties of specific complexes in mixed populations. 2. Materials and Methods (a) Materials RNA polymerase holoenzyme was purified from Escherichia coli DGI56 cells by the method of Gonzalez et al. (1977). The holoenzyme preparations used in these studies contained 40% to 65% active RNA polymerase molecules relative to the amount of protein added, as determined by the quantitative assay for RNA polymerases (Chamberlin et al., 1979). E. coli sigma factor was purified by the method of Gribskov & Burgess (1983) from the sigma-70 overproducing strain M5219 (pMRGS), which was kindly provided by R. Burgess. Sigma factor was assayed for its ability to stimulate transcription of phage T7 + DNA by purified core RNA polymerase (Berg et al., 1971). E. coli Nasa and NusB proteins were purified as described (Schmidt & Chamberlin, 1984; Schmidt, 1985). RNA polymerase, sigma factor, NusA protein, and NusB protein were diluted immediately before use by gradual addition of E. coli RNA polymerase diluent (Chamberlin et al., 1979). DNA templates were prepared as described (Levin et al., 1987) from T7 deletion phages (Studier et al.. 1979) and from plasmid pARl707 (Studier & Rosenberg, 1981; Briat & Chamberlin, 1984). DNA concentrations were determined by measuring the absorbance at 260 nm. To generate a linear transcription template, pAR1707 plasmid DNA was digested with the restriction endonuclease SalI (Bethesda Research Laboratories) as suggested by the manufacturer and then extracted extensively with a l : l mixture of phenol and chloroform/ isoamyl alcohol (24 : l, v/v), followed by extraction with ether. The extracted DNA was precipitated with ethanol and dissolved in l0 mM-Tris-HC1 (pH 8), l mM-EDTA. Unlabeled nucleoside triphosphates were purchased from Pharmacia PL Biochemicals and Boehringer Mannheim Biochemicals. Highly purified nucleoside triphosphates (Ultrapure grade) used in the synthesis of A20 complexes (see below) and nucleoside triphosphate derivatives methylated at the 3' position were purchased from Pharmacia PL Biochemicals. The dinucleotide ApU was purchased from Sigma. [a-32P]CTP was purchased from Amersham. Sephacryl S-300 (Superfine), Sephadex G-50 (Fine), and Sepharose CL-2B were purchased from Pharmacia. Acetylated bovine serum albumin was prepared by the method of Gonzalez et al. (1977). Single-stranded DNA oligonucleotides were synthesized by the solid-phase phosphoramidite method on a Biosearch 8750 DNA synthesizer. Full-length oligonucleotides were separated from shorter, incomplete chains by denaturing polyacrylamide gel electrophoresis and recovered from gel slices by diffusion into l0 mM-Tris'HCl (pH 8), 1 mM-EDTA. The oligonucleotides were concentrated by repeated extraction with n-butanol, precipitated with ethanol, and dissolved in t0 mM-Tris.HCI (pH 8), 1 mM-EDTA. Oligonucleotide concentrations were determined by measuring the absorbance at 260 nm. To determine the effect of the 3'-terminal RNA sequence on ternary complex stability at positions 37 to 39 of the T7Dlll A1 transcription unit, templates containing the T7 A1 promoter and the first 30n'f of the Abbreviations used: n, nucleotide(s); OAc, acetate ion; Glu, glutamate ion.

R N A Polymerase Complexes T 7 D l l I A1 transcription unit followed by 15n of random sequence were constructed. Two single-stranded DNA oligonucleotide preparations were used in the construction of these plasmid templates. One oligomer, corresponding to the non-transcribed template strand, encoded the AI promoter and the first 17n of the Dl I1 Al transcript. The other oligomer, corresponding to the transcribed template strand, encoded part of the A1 promoter, the first 30n of the D i l l Al transcript, and 15n of random sequence. The 3' ends of these oligomers were complementary over a region of 31 nucleotides. The 5' end of the non-transcribed strand oligomer encoded a B a m H I restriction site, and the 5' end of the transcribed strand oligomer encoded an EcoRI restriction site. The 2 complementary o]igomers (l pg of each) were mixed and incubated at 55°C for 30rain in 20pl of buffer (30 mi-Tris" HCl (pH 7"5), 150 mm-NaC1, 30 mm-MgC12, 15mm-dithiothreitol, 100pg acetylated bovine serum albumin/ml). The mixture was then allowed to cool slowly (30min) to room temperature. Deoxyribonucleoside triphosphates were added to a final concentration of 250 #M, and the final reaction volume was increased to 58 pl by the addition of water. Twelve units of DNA polymerase I Klenow fragment were added, and the reaction was incubated for 30 rain at room temperature. The filled-in fragments were extracted and concentrated as described above and then digested with restriction endonucleases B a m H I and EcoRI. Restricted fragments were inserted into the vector pUC119 (Vieira & Messing, 1987) which had been digested with BamHI and EcoRI. Standard procedures for subcloning were used. Sequencing of supercoiled plasmids by the chain termination method employed the Sequenase enzyme and was performed as suggested by the supplier, United States Biochemical Corporation (Cleveland, Ohio). Prior to use in transcription experiments, these plasmid templates were digested with HindIII. (b) Transcript release reactions To follow transcript release from ternary transcription complexes halted at many different template sites, the transcript release assay outlined in Fig. l was used, except where noted otherwise. Transcription initiation was limited to the T7 Al promoter and elongation was synchronized by forming ternary transcription complexes halted at position 20 (A20 complexes) on each of the templates studied (Levin et al., 1987). The conditions used to prepare A20 complexes allowed transcripts to be effectively end-labeled and assured that no more than one transcription complex was present per DNA template molecule (Levin et al., 1987). The procedure described by Levin el al. (1987) for preparing A20 complexes was used, except that the transcription buffer contained 44 mm-Tris-HCI (pH 8), 4 mM-MgC12, 14 mm-2-mercaptoethanol, 20 mr~-KCl, 2% (v/v) glycerol, 0-04 mm-EDTA, and 40 ~g acetylated bovine serum albumin/ml. RNA polymerase holoenzyme (total protein) and DNA templates were present at a concentration of 18 nM, except for experiments with D167 template in which 12 nm-DNA template and 18 nm-RNA polymerase were used. Re-initiation was prevented by the addition of 100 pg rifampicin/ml. A20 complexes (10/~l) were allowed to elongate at 30°C by addition of a standard reaction buffer (40/~l) to give final concentrations of l0 pm each of ATP, CTP, GTP and UTP, 40 mm-Tris" HCI (pH 8), 10 mM-2-mercaptoethanol, 4 mM-MgC12, 4 mmspermidine and 120 mm-KC1, unless indicated otherwise. Elongation was stopped after times ranging from 5 s to

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Figure 1. Release assay protocol. To limit initiation to the T7 Al promoter and to synchronize elongation, ternary complexes were formed at position 20 on each of the T7 templates (A20 complexes). Ternary complexes halted throughout the first 150 nucleotides of the transcription unit were prepared by addition of ATP, UTP, GTP and CTP (10 mm) to the A20 complexes, and elongation was stopped after times ranging from 5 to 90 s by addition of 20 mm-EDTA. EDTA-quenched complexes were pooled to generate a mixed population of paused ternary complexes. After various times of incubation at 30°C (release reaction), samples were withdrawn and mixed with a chase solution (usually 100/Ira each of ATP, GTP, CTP and UTP, 20mm-MgC12). After 10rain at 30°C, elongation was stopped and labeled RNA products were analyzed by gel electrophoresis as described in Materials and Methods.

90 s by addition of 220 mM-EDTA (5 ~1). These EDTAquenched ternary complexes were then pooled to generate a mixed population of complexes with polymerase paused at many different sites along the DNA template molecules. After the indicated times of incubation at 30°C, samples were withdrawn from the ternary complex pool and mixed with a "chase" solution so that the final reaction mixture contained 100 pm each of ATP, UTP, CTP and GTP and 20 mm-MgCl2, unless stated otherwise. Elongation during this chase period was allowed to proceed for l0 min at 30°C; this time is sufficient for the completion oii all RNA chains by stable ternary complexes. Two volumes of a stop solution (1"5 M-NH+OAc, 37"5 mM-EDTA, 50/ag tRNA/ml) were then added. Samples were precipitated with 2-5 vol. ethanol, resuspended in formamide loading buffer (80~/o (v/v) deionized formamide, 1 × TBE (89 mM-Tris-borate,

82

K. M . Arndt and M. J. Chamberlin

89mm-boric acid, 2mM-EDTA), 0"03% (w/v) xylene cyanol, 0.03% (w/v) bromphenol blue), and loaded on 15% (w/v) acrylamide/8"3 M-urea gels for analysis. This general protocol was modified for experiments employing the pAR1707 template (Fig. 2(g)) or high ionic strength conditions (Fig. 7). In these experiments, the chase solution contained 1 mM each of ATP, GTP, CTP and UTP and 40 mm-MgCl2 (or Mg(OAc)2, see the legend to (Fig. 7) to allow efficient elongation through strong pause sites and under conditions of high ionic strength. In addition, the chase period was allowed to continue for 20 min in experiments employing the pAR1707 template. Quantification of transcript release was carried out by densitometric scanning of autoradiograms of gels. Dissociation rates were calculated assuming that dissociation is a simple, first-order process. For many experiments semilog plots of the data were linear supporting this assumption. However, the data for some experiments gave plots that were non-linear, or the data scattered sufficiently to preclude accurate analysis. Such experiments usually involved sites at which release was very rapid; we did not try to take many time points to estimate these rates quantitatively. For such curves, a best fit of the early points through the origin was drawn to estimate the dissociation half-time. Transcript release from ternary complexes on the T 7 D l l l template were confirmed by a gel filtration assay. A mixture of EDTA-quenched D i l l ternary complexes was prepared as described above and incubated at 30°C for 60 min to allow transcript release to occur. A sample of this pool (200 #l) was then loaded onto a Sepharose CL-2B column (0"9 cm × 28 cm) equilibrated in 40 mm-Tris" HCI (pH 8), 120 mm-KCi, 4 mMspermidine, 10 mM-2-mercaptoethanol, and 20mMEDTA. The column was run at room temperature at a flow rate of 0-25 ml/min. Fractions (0"5 ml) were collected and assayed for trichloroacetic acid-precipitable radioactivity as described by Chamberlin et al. (1979) and for total radioactivity by spotting samples on glass fiber filters (Whatman GF/C filters), drying, and counting in a non-aqueous scintillation fiuor (Betamax, WestChem, San Diego). A sample (100 ~l) of selected fractions was mixed with a chase solution containing final concentrations of 1 mM each of ATP, UTP, GTP and CTP, 40 mm-MgCl2 and 100 #g rifampicin/ml, and subsequently incubated for l0 min at 30°C. These "chased" samples and samples (100/~l) taken directly from column fractions were quenched in stop solution and analyzed by gel electrophoresis as described above to distinguish fractions containing intact, stable ternary complexes from fractions containing free RNA. (c) R N A polymerase release assay To determine whether release of active RNA polymerase accompanies transcript release at sites of ternary complex instability, the standard transcript release assay (Fig. l) was modified as follows. T7D1 l l A20 complexes, were prepared as described above in a reaction containing 20 nM-RNA polymerase and 20 nM-template, and were treated with 100/~g rifampicin/ml to inactivate free and promoter-bound RNA polymerase molecules. Unbound rifampicin and unincorporated nucleoside triphosphates were removed from these rifampicin-treated A20 complexes by centrifugal gel filtration on a 1 ml column of Sephadex G-50 (Fine) (Levin et al., 1987). A "reporter template", plasmid pARI707, and purified E. coli RNA polymerase sigma-70 factor (both present at 92 mm or approx. 6"5-fold molar excess relative to the concentration

of A20 complexes) were added to the column-purified A20 complexes. As described for the standard assay, A20 complexes were then elongated at 30°C by addition of 10 pm each of ATP, UTP, GTP and CTP, and elongation was stopped after 5, 15 and 30s by addition of 20mM-EDTA. EDTA-quenched reactions were pooled and incubated at 30°C for the indicated "release" times. Samples were analyzed for transcript release from Dl I l as described above (Fig. 1), except that the 10 min chase reaction was carried out in the presence of 92/~m each of ATP, UTP, GTP and CTP, 20 mM-MgCi2, and 77 #g rifampicin/ml. A second sample of the ternary complex pool was assayed for transcription of the pARl707 reporter template by addition of 400 pm ATP, UTP, GTP and [a-32P]CTP (3500 cts/min per pmol), 20 mm-MgCl2, and 100#g rifampicin/ml. Transcription of the pARt707 template was allowed to continue for 10min but was restricted to a single round by the presence of rifampicin. Transcripts released from the DI 11 template were identified by etectrophoresis on a 15~/o acrylamide/8"3 M-urea gel, and transcripts produced on the pAR1707 template were analyzed on a 5~/o acrylamide/8"3m-urea gel as described above. Quantification of D l l l and pAR1707 transcripts was performed by excising appropriate gel slices and counting the dried slices in a non-aqueous scintillation fluor. Molar amounts of these transcripts were calculated by comparison with RNA standards of known specific activity that were also subjected to gel electrophoresis and scintillation counting. (d) Specific inhibition of transcript release by a complementary D N A oligomer Transcript release occurs very rapidly from a number of sites on the templates we studied under our standard conditions. To measure the effect of various factors on the rate of transcript release from these sites, we employed conditions that increase the stability of ternary transcription complexes paused at these sites. Complexes isolated by gel filtration chromatography in low ionic strength buffers dissociate more slowly than transcription complexes quenched with 20 mM-EDTA in our standard conditions, although the same sites of transcript release are observed. Thus, to determine the effect of a complementary DNA oligomer on transcript release at certain sites, addition of EDTA to stop chain elongation was avoided, and ternary complexes were separated from unincorporated nucleoside triphosphates by gel filtration chromatography. A20 complexes on the T 7 D l l l template were prepared and separated from unincorporated nucleoside triphosphates by centrifugal gel filtration on a l ml Sephadex G-50 (Fine) column as described above, and rifampicin was added to a final concentration of 100 pg/ml. The column-purified A20 complexes were elongated by addition of 100 nM each of ATP, UTP, GTP and CTP in 44 mm-Tris- HCI (pH 8), 20 mM-KCI, 4 mm-MgC12, 14 mm-2-mercaptoethanol, 2% glycerol, 0-04 mM-EDTA and 40 ~g aeetylated bovine serum albumin/ml. After 30 min of elongation at 30°C, these ternary complexes were separated from unincorporated nucleoside triphosphates on a Sephacryl S-300 column (0-7 cm × 13 em) equilibrated in this same buffer and run at room temperature. Column fractions (0"2ml) containing ternary complexes were identified by an ethidium bromide spot test and by scintillation counting samples. Fractions containing ternary complexes were pooled, and rifampicin was added to a final concentration of 100 ~g/ml. Ternary complexes (approx. 0"5rim) were then incubated for

R N A Polymerase Complexes 10min at 30°C with 10/~m-single-stranded DNA oligonucleotide or buffer (10 mM-Tris"HC1 (pH 8), 1 mm-EDTA). After this binding step, the KCI concentration in the reaction was increased to 120 mm to accelerate the transcript release step. At the indicated times after KCI addition, samples were chased for l0 min in the presence of 1 mM each of ATP, UTP, GTP and CTP. Samples were analyzed by gel electrophoresis as described above.

(e) Synthesis of RNA size markers Chain-terminating 3'-O-methyl nucleoside triphosphates were used to generate sequence-specific RNA size standards. A20 ternary complexes were allowed to elongate for l0 rain at 30°C in the presence of one 3'-O-methyl nucleoside triphosphate and the 4 unmodified substrates. In the presence of 10/~m each of ATP, GTP, CTP and UTP, the following concentrations of 3'-O-methyl substrates w e r e employed: A ladder, 0.2 mM3'-O-MeATP; U ladder, 0"2 mr~-3'-O-MeUTP; G ladder, 0"05 mML3'-O-MeGTP; C ladder, 0-04 mM-3'-O-MeCTP. (f) Sequence a~mlysis Computer analysis was used to predict RNA secondary structures. The sequence analysis package employed was based on the work of Zuker & Stiegler (1981) and was accessed through a SUN Microsystems work station. 3. R e s u l t s

(a) Rationale Our objective in these studies was to characterize the properties of a large number of ternary transcription complexes at different sites along the DNA template. We were particularly interested in how different sequences might affect the stability of such complexes to dissociation and transcript release. While methods are available for preparation of pure populations of specific ternary complexes, the procedures are laborious and time-consuming and not suitable for large-scale screening of many different sites (Levin et al., 1987; Kassavetis et al., 1986). However, we have devised a method to study certain properties of individual complexes even within a mixed population (Fig. 1). This approach involves the preparation of a mixed population of transcription complexes containing RNA polymerase molecules halted during normal elongation at many different sites. Under conditions where elongation is blocked, the dissociation rate of each of these complexes can be measured, since dissociation leads to release of an RNA of characteristic size. When the block to elongation is lifted, intact transcription complexes complete their transcripts while the RNAs released from unstable complexes remain. The rate of release can be followed quantitatively by RNA gel electrophoresis. We chose to study transcription complexes formed with several phage T7 deletion m u t a n t DNAs, since we have studied chain elongation through these sequences in great detail (Levin & Chamberlin, 1987). To avoid experimental difficul-

83

ties associated with promoter-binding and chain initiation, transcription complexes were first formed with each template under conditions that half transcription after 20n (A20 complexes: Levin et al., 1987) and assure only a single transcription complex per template. These A20 complexes were then used to prepare mixed populations of ternary complexes at sites further downstream. Elongation was usually halted with EDTA, and complexes were then incubated for varying times under different reaction conditions to allow dissociation. Transcription was resumed by addition of excess Mg2+, and the resulting population of RNAs was analyzed by highresolution gel electrophoresis.

(b) Ternary transcription complexes vary greatly in

their stability Using this approach, we have measured the stability of ternary transcription complexes on six different T7 deletion mutant templates and one plasmid template, pAR1707, which contains the T7 A1 promoter and part of the T7 early transcribed region. Ternary transcription complexes halted at most sites on these templates by the addition of EDTA are stable for more than two hours in the moderate ionic conditions of the standard assay; the transcripts associated with these complexes are completed when Mg2+ in excess of the EDTA is added back to the reaction (Fig. 2). Furthermore, the pattern of ternary complex stability on these templates does not change markedly even after extended times of incubation at 30°C; the vast majority of halted complexes can resume elongation after 24 hours under the standard assay conditions (Fig. 3). We will refer to these very stable ternary complexes as normal complexes. In contrast to the extreme stability of most complexes, ternary complexes at a number of specific sites are very unstable, as judged by their inability to resume elongation (Fig. 2). We show in subsequent sections that, for the majority of these sites, the inability to resume elongation is due to dissociation of RNA polymerase and RNA from the template, and we will refer to these as release sites. The kinetics of ternary complex dissociation at such loci vary from site to site. For the seven T7 templates shown in Figure 2, we have identified at least nine different sites at which complexes have life-times of less than two hours in the standard transcription buffer containing 20 mM-EDTA. The template sequences at each of these sites are shown in Figure 4. With the exception of T7 deletion template D123, all of the templates studied have the same sequence up to nucleotide 30 of the A1 transcript and display identical patterns of ternary complex stability within this region. In addition, the A1 transcripts of D104, D l l 2 , and D387 are identical up to nucleotide 79, and ternary complexes halted within this conserved region have similar properties. An obvious site of complex instability for all of

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